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At least 289 records · Page 16

Expression of the genes for insulin-like growth factors and their receptors in bone during skeletal growth

Insulin-like growth factors (IGF) are important regulators of skeletal growth. To determine whether the capacity to produce and respond to these growth factors changes during skeletal development, we measured the protein and mRNA levels for IGF-I, IGF-II, and their receptors (IGF-IR and IGF-IIR, respectively) in the tibia and femur of rats before and up to 28 mo after birth. The mRNA levels remained high during fetal development but fell after birth, reaching a nadir by 3-6 wk. This fall was most pronounced for IGF-II and IGF-IIR mRNA and least pronounced for IGF-I mRNA. However, after 6 wk, both IGF-I and IGF-IR mRNA levels recovered toward the levels observed at birth. In the prenatal bones, the signals for the mRNAs of IGF-II and IGF-IIR were stronger than the signals for the mRNAs of IGF-I and IGF-IR, although the content of IGF-I was three- to fivefold greater than that of IGF-II. IGF-II levels fell postnatally, whereas the IGF-I content rose after birth such that the ratio IGF-I/IGF-II continued to increase with age. We conclude that, during development, rat bone changes its capacity to produce and respond to IGFs with a progressive trend toward the dominance of IGF-I.

NASA Discipline Musculoskeletal↗

Cytokinin signaling localized in phloem noncell‐autonomously regulates cambial activity during secondary growth of Populus stems

Summary The regulation of cytokinin on secondary vascular development has been uncovered by modulating cytokinin content. However, it remains unclear how cytokinin enriched in developing secondary phloem regulates cambium activity in poplar. Here, we visualized the gradient distribution of cytokinin with a peak in the secondary phloem of poplar stem via immunohistochemical imaging, and determined the role of phloem‐located cytokinin signaling during wood formation. We generated transgenic poplar harboring cytokinin oxidase/dehydrogenase ( CKX)2 , a gene encoding a cytokinin degrading enzyme, driven by the phloem‐specific CLE41b promoter, indicating that the disruption of the cytokinin gradient pattern restricts the cambial activity. The RNA interference‐based knockdown of the histidine kinase ( HK ) genes encoding cytokinin receptors specifically in secondary phloem significantly compromised the division activity of cambial cells, whereas the phloem‐specific expression of a type‐B response regulator (RR) transcription factor stimulated cambial proliferation, providing evidence for the noncell‐autonomous regulation of local cytokinin signaling on the cambial activity. Moreover, the cambium‐specific knockdown of HK s also led to restricted cambial activity, and the defects were aggravated by the reduced cytokinin accumulation. Our results showed that local cytokinin signaling in secondary phloem regulates cambial activity noncell‐autonomously, and coordinately with its local signaling in cambium.

Fu, Xiaokang↗

Biological potential of extraterrestrial materials. 2. Microbial and plant responses to nutrients in the Murchison carbonaceous meteorite

Meteoritic materials are investigated as potential early planetary nutrients. Aqueous extracts of the Murchison C2 carbonaceous meteorite are utilized as a sole carbon source by microorganisms, as demonstrated by the genetically modified Pseudomonas fluorescence equipped with the lux gene. Nutrient effects are observed also with the soil microorganisms Nocardia asteroides and Arthrobacter pascens that reach populations up to 5 x 10(7) CFU/ml in meteorite extracts, similar to populations in terrestrial soil extracts. Plant tissue cultures of Asparagus officinalis and Solanum tuberosum (potato) exhibit enhanced pigmentation and some enhanced growth when meteorite extracts are added to partial nutrient media, but inhibited growth when added to full nutrient solution. The meteorite extracts lead to large increases in S, Ca, Mg, and Fe plant tissue contents as shown by X-ray fluorescence, while P, K, and Cl contents show mixed effects. In both microbiological and plant tissue experiments, the nutrient and inhibitory effects appear to be best balanced for growth at about 1:20 (extracted solid : H2O) ratios. The results suggest that solutions in cavities in meteorites can provide efficient concentrated biogenic and early nutrient environments, including high phosphate levels, which may be the limiting nutrient. The results also suggest that carbonaceous asteroid resources can sustain soil microbial activity and provide essential macronutrients for future space-based ecosystems.

Non-NASA Center↗

Expression of blue pigment synthetase a from Streptomyces lavenduale reveals insights on the effects of refactoring biosynthetic megasynthases for heterologous expression in Escherichia coli .

High GC bacteria from the genus Streptomyces harbor expansive secondary metabolism. The expression of biosynthetic proteins and the characterization and identification of biological "parts" for synthetic biology purposes from such pathways are of interest. However, the high GC content of proteins from actinomycetes in addition to the large size and multi-domain architecture of many biosynthetic proteins (such as non-ribosomal peptide synthetases; NRPSs, and polyketide synthases; PKSs often called "megasynthases") often presents issues with full-length translation and folding. Here we evaluate a non-ribosomal peptide synthetase (NRPS) from Streptomyces lavenduale, a multidomain "megasynthase" gene that comes from a high GC (72.5%) genome. While a preliminary step in revealing differences, to our knowledge this presents the first head-to-head comparison of codon-optimized sequences versus a native sequence of proteins of streptomycete origin heterologously expressed in E. coli. We found that any disruption in co-translational folding from codon mismatch that reduces the titer of indigoidine is explainable via the formation of more inclusion bodies as opposed to compromising folding or posttranslational modification in the soluble fraction. In conclusion, this result supports that one could apply any refactoring strategies that improve soluble expression in E. coli without concern that the protein that reaches the soluble fraction is differentially folded.

59 BASIC BIOLOGICAL SCIENCES↗

Pseudodesulfovibrio mercurii sp. nov., a mercury-methylating bacterium isolated from sediment

The sulfate-reducing, mercury-methylating strain ND132 T was isolated from the brackish anaerobic bottom sediments of Chesapeake Bay, USA. Capable of high levels of mercury (Hg) methylation, ND132 T has been widely used as a model strain to study the process and to determine the genetic basis of Hg methylation. Originally called Desulfovibrio desulfuricans ND132 T on the basis of an early partial 16S rRNA sequence, the strain has never been formally described. Phylogenetic and physiological traits place this strain within the genus Pseudodesulfovibrio, in the recently reclassified phylum Desulfobacterota (formerly Deltaproteobacteria ). ND132 T is most closely related to Pseudodesulfovibrio hydrargyri BerOc1 T and Pseudodesulfovibrio indicus J2T. Analysis of average nucleotide identity (ANI) of whole-genome sequences showed roughly 88 % ANI between P. hydrargyri BerOc1T and ND132T, and 84 % similarity between ND132T and P. indicus J2 T . These cut-off scores <95 %, along with a multi-gene phylogenetic analysis of members of the family Desulfovibrionacea, and differences in physiology indicate that all three strains represent separate species. The Gram-stain-negative cells are vibrio-shaped, motile and not sporulated. ND132 T is a salt-tolerant mesophile with optimal growth in the laboratory at 32 °C, 2 % salinity, and pH 7.8. The DNA G+C content of the genomic DNA is 65.2 %. It is an incomplete oxidizer of short chain fatty acids, using lactate, pyruvate and fumarate with sulfate or sulfite as the terminal electron acceptors. ND132 T can respire fumarate using pyruvate as an electron donor. The major fatty acids are iso-C 15 :0 , anteiso-C 15 : 0 , iso-C 17 : 0 , iso-C 17 : 1 ω9c and anteiso-C 17 : 0 . We propose the classification of strain ND132 T (DSM 110689, ATCC TSD-224) as the type strain Pseudodesulfovibrio mercurii sp. nov.

59 BASIC BIOLOGICAL SCIENCES↗

Grape ASR Regulates Glucose Transport, Metabolism and Signaling

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

59 BASIC BIOLOGICAL SCIENCES↗

Post-Synthetic Reduction of Pectin Methylesterification Causes Morphological Abnormalities and Alterations to Stress Response in Arabidopsis thaliana

Pectin is a critical component of the plant cell wall, supporting wall biomechanics and contributing to cell wall signaling in response to stress. The plant cell carefully regulates pectin methylesterification with endogenous pectin methylesterases (PMEs) and their inhibitors (PMEIs) to promote growth and protect against pathogens. We expressed Aspergillus nidulans pectin methylesterase (AnPME) in Arabidopsis thaliana plants to determine the impacts of methylesterification status on pectin function. Plants expressing AnPME had a roughly 50% reduction in methylester content compared with control plants. AnPME plants displayed a severe dwarf phenotype, including small, bushy rosettes and shorter roots. This phenotype was caused by a reduction in cell elongation. Cell wall composition was altered in AnPME plants, with significantly more arabinose and significantly less galacturonic acid, suggesting that plants actively monitor and compensate for altered pectin content. Cell walls of AnPME plants were more readily degraded by polygalacturonase (PG) alone but were less susceptible to treatment with a mixture of PG and PME. AnPME plants were insensitive to osmotic stress, and their susceptibility to Botrytis cinerea was comparable to wild type plants despite their compromised cell walls. This is likely due to upregulated expression of defense response genes observed in AnPME plants. These results demonstrate the importance of pectin in both normal growth and development, and in response to biotic and abiotic stresses.

59 BASIC BIOLOGICAL SCIENCES↗

Comparative genomics of the Liberibacter genus reveals widespread diversity in genomic content and positive selection history

‘Candidatus Liberibacter’ is a group of bacterial species that are obligate intracellular plant pathogens and cause Huanglongbing disease of citrus trees and Zebra Chip in potatoes. Here, we examined the extent of intra- and interspecific genetic diversity across the genus using comparative genomics. Our approach examined a wide set of Liberibacter genome sequences including five pathogenic species and one species not known to cause disease. By performing comparative genomics analyses, we sought to understand the evolutionary history of this genus and to identify genes or genome regions that may affect pathogenicity. With a set of 52 genomes, we performed comparative genomics, measured genome rearrangement, and completed statistical tests of positive selection. We explored markers of genetic diversity across the genus, such as average nucleotide identity across the whole genome. These analyses revealed the highest intraspecific diversity amongst the ‘Ca. Liberibacter solanacearum’ species, which also has the largest plant host range. We identified sets of core and accessory genes across the genus and within each species and measured the ratio of nonsynonymous to synonymous mutations (dN/dS) across genes. We identified ten genes with evidence of a history of positive selection in the Liberibacter genus, including genes in the Tad complex, which have been previously implicated as being highly divergent in the ‘Ca. L. capsica’ species based on high values of dN.

59 BASIC BIOLOGICAL SCIENCES↗

Disruption of Brachypodium lichenase alters metabolism of mixed‐linkage glucan and starch

Mixed-linkage glucan, which is widely distributed in grasses, is a polysaccharide highly abundant in cell walls of grass endosperm and young vegetative tissues. Lichenases are enzymes that hydrolyze mixed-linkage glucan first identified in mixed-linkage glucan-rich lichens. In this study, we identify a gene encoding a lichenase we name Brachypodium distachyon LICHENASE 1 (BdLCH1), which is highly expressed in the endosperm of germinating seeds and coleoptiles and at lower amounts in mature shoots. RNA in situ hybridization showed that BdLCH1 is primarily expressed in chlorenchyma cells of mature leaves and internodes. Disruption of BdLCH1 resulted in an eight-fold increase in mixed-linkage glucan content in senesced leaves. Consistent with the in situ hybridization data, immunolocalization results showed that mixed-linkage glucan was not removed in chlorenchyma cells of lch1 mutants as it was in wild type and implicate the BdLCH1 enzyme in removing mixed-linkage glucan in chlorenchyma cells in mature vegetative tissues. We also show that mixed-linkage glucan accumulation in lch1 mutants was resistant to dark-induced degradation, and 8-week-old lch1 plants showed a faster rate of starch breakdown than wild type in darkness. Here our results suggest a role for BdLCH1 in modifying the cell wall to support highly metabolically active cells.

59 BASIC BIOLOGICAL SCIENCES↗

Planetary Protection at Marshall Space Flight Center

Introduction: NASA Marshall Space Flight Center (MSFC) is historically known for its role in propulsion. While this is still the mainstay of MSFC’s expertise, many unique capabilities exist at MSFC which pertain to Planetary Protection (PP), including 1) identifying PP threats, and 2) developing novel methods to neutralize those threats. Furthermore, because these capabilities exist among diverse groups at MSFC, this work promotes collaboration both within and outside MSFC to expand and develop PP studies related to a full spectrum of NASA research, design, manufacture, and test interests. This abstract describes the PP research ongoing at MSFC and describes how it contributes to NASA’s overall PP objectives. Microbial Identification in Cleanrooms: One of the greatest threats to successful implementation of PP requirements is recontamination post bioburden reduction. One method to prevent recontamination is to keep the spacecraft in clean environments (i.e. cleanrooms) as much as possible during assembly and integration. However, cleanrooms are not without their own sources of contamination, which is why NASA is interested in monitoring the cleanliness of cleanrooms and characterizing the microbial species present. Such information allows a greater understanding of the resistance of these microbes to cleaning methods, as well as the risk of their contaminating the targeted planetary body of a given mission. MSFC has multiple cleanrooms of various ISO cleanliness levels onsite. We sampled the air and surfaces of three of these rooms, isolated microbes, and then sequenced the 16S rRNA gene or ITS region of the 18S rRNA gene for bacterial and fungal isolates, respectively. This has resulted in a microbial library which currently includes nearly 100 isolates. Microbial Enumeration of Spacecraft Materials: Currently, there are only a couple bioburden reduction methods approved by NASA, and often the harshness of these methods presents additional concerns or risks related to material properties. The goal of this research is to assess the microbial content of solid rocket motor (SRM) materials potentially used for lander missions. This work aims to more accurately define the risk of planetary contamination by providing empirical data associated with commonly used SRM raw materials. In this study, we pulverized nonmetallic SRM materials using a cryogenic grinder, then analyzed the resulting substrate for microbial colony forming units (CFU). We found that many SRM nonmetallic materials do not harbor detectable bioburden, though a range existed depending on the material. The results from this work provide quantitative data to potentially reduce concerns of contamination, while also providing a foundation for follow up studies into additional sterilization methods and molecular identification of contaminating microbes. Space Environmental Effects on Microbial Survival: One potential area of microbial reduction is the space environment. Understanding the survivability of hardy microbes in space-like conditions is a crucial first step in answering how space may reduce bioburden and if it can be relied upon for adherence to PP requirements. This work studied the effects of ultraviolet (UV) and ionizing radiation on survival of Bacillus atrophaeus spores. Microbes were dried on relevant polymeric materials then exposed to space environmental stressors. Coupons were submerged in water, diluted, and plated to determine survival compared with controls. We found that both UV and ionizing radiation were capable of reducing viability by nearly 99%, but there were still survivors, some with changed morphology indicating resistance mechanisms within certain cells. Manufacturing credit: Finally, given the above-mentioned limitations of the NASA-approved bioburden reduction methods, there is interest in understanding if manufacturing processes may provide enough bioburden reduction without additional PP-specific bakeouts. For instance, some material additives may be antimicrobial. Given this, we investigated the effects of several commonly used rubber additives on the growth of B. atrophaeus spores. We found that some of the materials inhibited growth of the spores, possibly supporting the use of these additives on missions with PP constraints. Future work into manufacturing credit for bioburden reduction includes inoculation of green insulation with B. atrophaeus spores, followed by a typical cure. Thermal profiles will be verified for appropriate temperature and durations to meet PP requirements, and cured samples will be analyzed using a cryogenic grinder to determine survivability of spores.

Chelsi D. Cassilly↗

Synchrotron fluorescence imaging of individual mouse beta-cells reveals changes in zinc, calcium, and iron in a model of low-grade inflammation

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Here, primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

59 BASIC BIOLOGICAL SCIENCES↗

Cinnamon essential oil vapor alleviates the reduction of aroma-related volatiles in cold-stored “Feicheng” peach using HS-GC-IMS

“Feicheng” peach is popular for its unique aroma, but its defect of being highly sensitive to chilling injury (CI) often leads to aroma loss and internal browning. Essential oils (EOs) are often used to enhance the antioxidant capacity of plants and fruits, as well as to trigger their defense against biotic/abiotic stresses. This study aimed to examine the effect of cinnamon essential oil (CEO) vapor treatment on the aroma quality of peach fruit during cold storage using HS-GC-IMS. The results showed that 50 μL/L CEO vapor reduced the severity of internal browning (IB) in peaches at the stage of 7 ~ 21 d during refrigeration (Significantly, the L * value was higher and the IB index was lower than that of control, p < 0.05). Meanwhile, the evident reduction or loss of aroma content caused by CI was restored to a higher level than the control ( p < 0.05). Furthermore, CEO treatment promoted the release of aroma-related volatiles as evidenced by more propyl acetate, and the dimer of amyl acetate, isoamyl acetate, butyl acetate detected than that on harvest day and no-treated group after 21 d of cold storage plus 2 d of shelf life. Genes of PpLOX1 , PpLOX2 , PpHPL1 and PpADH1 associated with aroma-related volatile biosynthesis revealed higher transcript abundance in peach fruits treated with CEO than the control ( p < 0.05). Overall, our study demonstrated that CEO in vapor phase may be beneficial to alleviate the quality deterioration in aroma and flesh color of “Feicheng” peaches caused by CI, which lays a theoretical reference for maintaining postharvest quality of peach fruits.

Wang, Dan↗

Effects of gene deletion of the tissue inhibitor of the matrix metalloproteinase-type 1 (TIMP-1) on left ventricular geometry and function in mice

Alterations in the expression and activity of the matrix metalloproteinases (MMPs) and the tissue inhibitors of the MMPs (TIMPs) have been implicated in tissue remodeling in a number of disease states. One of the better characterized TIMPs, TIMP-1, has been shown to bind to active MMPs and to regulate the MMP activational process. The goal of this study was to determine whether deletion of the TIMP-1 gene in mice, which in turn would remove TIMP-1 expression in LV myocardium, would produce time-dependent effects on LV geometry and function. Age-matched sibling mice (129Sv) deficient in the TIMP-1 gene (TIMP-1 knock-out (TIMP-1 KO), n=10) and wild-type mice (n=10) underwent comparative echocardiographic studies at 1 and 4 months of age. LV catheterization studies were performed at 4 months and the LV harvested for histomorphometric studies. LV end-diastolic volume and mass increased (18+/-4 and 38+/-3%, respectively, P<0.05) at 4 months in the TIMP-1 KO group; a significant increase compared to wild-type controls (P<0.05). At 4 months, LV and end-diastolic wall stress was increased by over two-fold in the TIMP-1 KO compared to wild type (P<0.05). However, LV systolic pressure and ejection performance were unchanged in the two groups of mice. LV myocyte cross-sectional area was unchanged in the TIMP-1 KO mice compared to controls, but myocardial fibrillar collagen content was reduced. Changes in LV geometry occurred in TIMP-1 deficient mice and these results suggest that constitutive TIMP-1 expression participates in the maintenance of normal LV myocardial structure. Copyright 2000 Academic Press.

Non-NASA Center↗

Genomic Analysis of the Natural Variation of Fatty Acid Composition in Seed Oils of Camelina sativa

Camelina sativa is an oilseed crop that has shown strong promise as a biofuel feedstock. The profile of fatty acids greatly influences the oil quality; however, genetic mechanisms that determine the natural variation of fatty acid composition in camelina are not fully understood. A genome wide association study (GWAS) was performed to uncover genetic loci that may contribute to the contents of major fatty acids such as oleic and linolenic acids in camelina seed. Two approaches were taken to improve the GWAS efficiency. First, growing a diversity panel of 212 accessions in four locations and two nitrogen fertilization conditions revealed great variation in fatty acid contents in seeds. Second, using an improved reference genome, abundant markers, including 203,320 single nucleotide polymorphisms (SNPs) and 99,067 insertions/deletions (indels), were developed, which refined the population structure of the diversity panel. GWAS resulted in 118 genetic markers across 31 trait/treatment conditions. Closely linked markers were determined based on linkage decay and by comparing secondarily associated markers when highly associated ones were removed. Candidate genes were examined by comparing the pangenomes of 12 high-quality reference genomes. This study provides new resources to understand seed lipid metabolism and improve camelina oils through molecular breeding.

Life Sciences & Biomedicine - Other Topics↗

Geochemical and microbial characteristics of seepage water and mineral precipitates in a radwaste disposal facility impacted by seawater intrusion and high alkalinity

The construction of an underground facility can dramatically change the quality, flow direction, and level of groundwater. It may also impact subsurface microbial composition and activity. Groundwater quality was monitored over eight years in two observational wells near an underground disposal facility on the east coast of South Korea. The results showed dramatic increases in dissolved ions such as O 2 , Na, Ca, Mg, and SO 4 during facility construction. Seepage water samples downgradient from the silos and tunnels, and precipitates deposited along the seepage water flow path were collected to determine the impact inside the disposal facility. X-ray analysis (powder X-ray diffraction (pXRD) and X-ray absorption fine structure (XAFS)) were used to characterize the mineral precipitates. Microbial community composition was determined by 16S rRNA gene sequencing. The seepage water composition was of two types: Ca-Cl and Ca-Na-HCO 3 . The ratio of Cl and δ 18 O showed that the Ca-Cl type seepage water was influenced by groundwater mixed with seawater ranging from 2.7% to 15.1%. Various sulfate-reducing bacteria were identified in the Ca-Cl type seepage water, exhibiting relatively high sulfate content from seawater intrusion. Samples from the Ca-Na-HCO 3 type seepage water had an extremely high pH (>10) and abundance of Hydrogenophaga. The precipitates observed along the flow path of the seepage water included calcite, ferrihydrite, green rust, and siderite, depending on seepage water chemistry and microbial activity. This study suggests that the construction of underground structures creates distinct, localized geochemical conditions (e.g., high alkalinity, high salinity, and oxic conditions), which may impact microbial communities. These biogeochemical changes may have undesirable large-scale impacts such as water pump clogging. Overall, an understanding of the process and long-term monitoring are essential to assess the safety of underground facilities.

54 ENVIRONMENTAL SCIENCES↗

The first intron and promoter of Arabidopsis DIACYLGLYCEROL ACYLTRANSFERASE 1 exert synergistic effects on pollen and embryo lipid accumulation

Summary Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. In Arabidopsis , two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in a dgat1‐1/pdat1‐2 double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified. We transformed a dgat1‐1/dgat1‐1//PDAT1/pdat1‐2 parent with transgenic constructs containing the Arabidopsis DGAT1 promoter fused to the AtDGAT1 open reading frame either with or without the first intron. Triple homozygous plants were obtained, however, in the absence of the DGAT1 first intron anthers fail to fill with pollen, seed yield is c . 10% of wild‐type, seed oil content remains reduced (similar to dgat1‐1/dgat1‐1 ), and non‐Mendelian segregation of the PDAT1/pdat1‐2 locus occurs. Whereas plants expressing the AtDGAT1pro:AtDGAT1 transgene containing the first intron mostly recover phenotypes to wild‐type. This study establishes that a combination of the promoter and first intron of AtDGAT1 provides the proper context for temporal and tissue‐specific expression of AtDGAT1 in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes in Arabidopsis .

McGuire, Sean T.↗

Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway

Abstract Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

59 BASIC BIOLOGICAL SCIENCES↗

Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production

Abstract The aviation industry’s growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multiomics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to 3 d earlier at 10 °C. However, these alterations led to a tradeoff between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.

Rasoul, Amira (ORCID:0009000714290175)↗